Method for treating parathyroid disorders

ABSTRACT

A method for treating hypoparathyroidism and/or hypocalcemia by local administration of a neurotoxin, such as a botulinum toxin, to a parathyroid gland, thereby reducing an inhibitory effect upon parathyroid hormone secretion. A method for treating hyperparathyroidism and/or hypercalcemia by local administration of a neurotoxin, such as a botulinum toxin, to a sympathetic ganglion which innervates a parathyroid hormone secreting parathyroid cell, thereby reducing a stimulatory effect upon parathyroid hormone secretion.

BACKGROUND

[0001] The present invention relates to methods for treating parathyroiddisorders. In particular the present invention relates to methods fortreating parathyroid disorders by administration of a neurotoxin to apatient.

[0002] The adult human typically has four small parathyroid glands, eachweighing about 30 to 40 mg, located near the thyroid. The chief cells ofthe parathyroid glands can make and release parathyroid hormone (PTH),which functions to help maintain serum calcium homeostasis. Parathyroidhormone increases blood calcium level while calcitonin from the thyroidC cells acts to lower it.

[0003] Disorders of the parathyroid glands include hyperparathyroidismand hypoparathyroidism. Primary hyperparathyroidism is about twice asprevalent in females as it is in males, and this ratio increases withage. About 1 in 500 females over age of 40 and 1 in 2000 males over theage of 40 has primary hyperparathyroidism. In the United States about250,000 persons are afflicted with primary hyperparathyroidism.

[0004] Primary hyperparathyroidism exists when a disorder of parathyroidtissue itself, as the primary defect, results in the release into thecirculation of too much parathyroid hormone. Among the known causes ofprimary hyperparathyroidism are parathyroid adenoma, hyperplasia andcarcinoma. Secondary hyperparathyroidism is a reactive parathyroidhyperplasic phenomenon, which can accompany renal failure. Symptoms ofhyperparathyroidism can include nephrolithiasis, bone disease, pepticulcer, fatigue and hypertension.

[0005] Untreated hyperparathyroidism can result in the loss ofconsiderable amounts of bone mass due to the hypercalcemia which arisesfrom an excessive level of circulating parathyroid hormone. Thus a highlevel of parathyroid hormone causes osteoclastic bone reabsorption whichcan lead to multiple foci of bone destruction, osteitis fibrosa cysticaor von Recklinghausen's disease of bone.

[0006] Production of parathyroid hormone by the chief cells of theparathyroid glands is apparently regulated to a significant extent inthe normal parathyroid by both free calcium concentration inextracellular fluid and by levels of 1,25 dihydroxyvitamin D(calcitriol). Parathyroid hormone is a single chain, 84 amino acidresidue polypeptide which acts upon osteocytes and osteoclasts toincrease is the rate of release of calcium from blood into bone,apparently by stimulation of osteocytic osteolysis.

[0007] The treatment of choice for primary hyperparathyroidism issurgery to remove all or most of the hyperactive parathyroid tissue.Thallium-technetium subtraction scans, ultrasound, selective venoussampling, CT, MRI, and arteriography have been used to localize aparathyroid disorder. Unfortunately, it has been reported that in aboutone third of parathyroidectomies, surgery fails to cure thehyperparathyroidism because of surgical ineptness to remove theappropriate tissues. Furthermore, excessive removal of parathyroidglands tissue can cause tetany. Complications of parathyroidectomy caninclude hematoma, vocal cord paralysis, hypocalcemia, and persistenthypercalcemia. Thus, after parathyroidectomy 5% of patients havepermanent hypocalcemia, which therefore requires daily oralsupplementation or reimplantation of cryopreserved parathyroid tissue.

[0008] Significantly, while parathyroid adenoma can be treated byremoval of the one abnormal parathyroid gland, removal of multipleparathyroid glands is typically required to treat parathyroidhyperplasia. Furthermore, the cause or causes of primary parathyroidhyperplasia are unknown.

[0009] Alternates to surgery for primary hyperparathyroidism includeethanol block and embolization. Block by ethanol injection destroys theparathyroid gland or glands injected and can cause Horner's syndrome andvocal cord paralysis. Additionally, embolization to the artery supplyingan abnormal parathyroid gland while sometimes successful to infarct theparathyroid gland and normalize calcium levels, is a difficult procedurewith a limited success rate.

[0010] Primary hypoparathyroidism due to deficient PTH secretion cancause a low serum calcium due to a lack of PTH mediated bone resorptionand calcium reabsorption by the kidneys. Symptoms of hypocalcemiainclude neuromuscular irritability and tetany. Intravenous calcium isthe treatment of choice for primary hypoparathyroidism. Notably, PTHreplacement has also been used to treat primary hypoparathyroidism.Drawbacks to PTH replacement include lack of clinical experience, itmust be given by injection and it is expensive.

[0011] Parathyroid Innervation

[0012] With regard to parathyroid innervation, one view is that thenerves to the parathyroids are only vasomotor, not secretomotor innature, and that parathyroid activity is controlled solely by variationin blood calcium level. Thus, a rise in blood calcium level inhibits PTHrelease, while a fall in blood calcium level stimulates PTH release.

[0013] Significantly it has been reported that parasympatheticinfluences inhibit parathyroid hormone secretion, that cholinergicagonists decrease serum PTH and that this effect is blocked by atropine.See e.g. J. Auto Nerv Syst 1994;48:45-53, Metabolism 1985;34(7):612-615and Brazilian J Med Biol Res 1994;27:573-599.

[0014] Additionally, the close anatomic association of the thyroid andparathyroid glands makes it reasonable to assume that the parathyroidsare innervated in a manner similar to the thyroid. The two upperparathyroid glands are located adjacent to the posterior surface of theupper or, middle part of the thyroid lobe, often just anterior to therecurrent laryngeal nerve as it enters the larynx. The two lowerparathyroid glands are usually found on the lateral or posteriorsurfaces of the lower part of the thyroid gland or within severalcentimeters of the lower thyroid pole within the thymic tongue.

[0015] It is known that the thyroid gland receives innervation from boththe sympathetic and parasympathetic divisions of the autonomic nervoussystem. The sympathetic fibers arise from the cervical ganglia and enterwith blood vessels, whereas the parasympathetic fibers are derived fromthe vagus and reach the gland via branches of the laryngeal nerves. Thethyroid gland's relation to the recurrent laryngeal nerves and to theexternal branch of the superior laryngeal nerves is of major surgicalsignificance, since damage to these nerves can lead to a disability ofphonation.

[0016] Sympathetic innervation of the thyroid cells has been reported toexert a stimulatory effect upon thyroid hormone release throughadrenergic receptors for norepinephrine on follicle cells. Endocrinology1979;105:7-9. Significantly, the human thyroid is also innervated bycholinergic, parasympathetic fibers. Cell Tiss Res 1978;195:367-370. Seealso Biol Signals 1994;3:15-25. And other mammalian species are known toalso have cholinergicly innervated thyroid cells. See e.g. Z. MikroskAnat Forsch Leipzig 1986;100:1,S, 34-38 (pig thyroid is cholinergiclyinnervated); Neuroendocrinology 1991;53:69-74 (rat thyroid ischolinergicly innervated); Endocrinology 1984;114:1266-1271 (dog thyroidis cholinergicly innervated);

[0017] Significantly, the consensus is that cholinergic, parasympatheticinfluence upon thyroid hormone secretion by thyroid follicle cells ininhibitory. Endocrinology 1979;105:7-9; Endocrinology1984;114:1266-1271; Peptides 1985;6:585-589; Peptides 1987;8:893-897,and; Brazilian J Med Biol Res 1994;27:573-599. The direct cholinergicinfluence upon the thyroid appears to be mediated by muscarinicacetylcholine receptors of thyroid follicle cells since the cholinergicinhibition is blocked by atropine. Endocrinology 1979;105:7.

[0018] Thus, one can conclude that, at least in some circumstances, thedeficient PTH secretion of primary hypoparathyroidism is influenced byinhibitory parasympathetic innervation of the parathyroids, whileprimary parathyroid hyperplasia is influenced by excessive sympatheticstimulation of the parathyroids.

[0019] Botulinum Toxin

[0020] The anaerobic, gram positive bacterium Clostridium botulinumproduces a potent polypeptide neurotoxin, botulinum toxin, which causesa neuroparalytic illness in humans and animals referred to as botulism.The spores of Clostridium botulinum are found in soil and can grow inimproperly sterilized and sealed food containers of home basedcanneries, which are the cause of many of the cases of botulism. Theeffects of botulism typically appear 18 to 36 hours after eating thefoodstuffs infected with a Clostridium botulinum culture or spores. Thebotulinum toxin can apparently pass unattenuated through the lining ofthe gut and attack peripheral motor neurons. Symptoms of botulinum toxinintoxication can progress from difficulty walking, swallowing, andspeaking to paralysis of the respiratory muscles and death.

[0021] Botulinum toxin type A is the most lethal natural biologicalagent known to man. About 50 picograms of a commercially availablebotulinum toxin type A (purified neurotoxin complex)¹ is a LD₅₀ in mice(i.e. 1 unit). Interestingly, on a molar basis, botulinum toxin type Ais about 1.8 billion times more lethal than diphtheria, about 600million times more lethal than sodium cyanide, about 30 million timesmore lethal than cobra toxin and about 12 million times more lethal thancholera. Singh, Critical Aspects of Bacterial Protein Toxins, pages63-84 (chapter 4) of Natural Toxins II, edited by B. R. Singh et al.,Plenum Press, New York (1976) (where the stated LD₅₀ of botulinum toxintype A of 0.3 ng equals 1 U is corrected for the fact that about 0.05 ngof BOTOX® equals 1 unit). One unit (U) of botulinum toxin is defined asthe LD₅₀ upon intraperitoneal injection into female Swiss Webster miceweighing 18 to 20 grams each.

[0022] Seven immunologically distinct botulinum neurotoxins have beencharacterized, these being respectively botulinum neurotoxin serotypesA, B, C₁, D, E, F and G each of which is distinguished by neutralizationwith type-specific antibodies. The different serotypes of botulinumtoxin vary in the animal species that they affect and in the severityand duration of the paralysis they evoke. For example, it has beendetermined that botulinum toxin type A is 500 times more potent, asmeasured by the rate of paralysis produced in the rat, than is botulinumtoxin type B. Additionally, botulinum toxin type B has been determinedto be non-toxic in primates at a dose of 480 U/kg which is about 12times the primate LD₅₀ for botulinum toxin type A. Botulinum toxinapparently binds with high affinity to cholinergic motor neurons, istranslocated into the neuron and blocks the release of acetylcholine.

[0023] Botulinum toxins have been used in clinical settings for thetreatment of neuromuscular disorders characterized by hyperactiveskeletal muscles. Botulinum toxin type A has been approved by the U.S.Food and Drug Administration for the treatment of blepharospasm,strabismus and hemifacial spasm. Non-type A botulinum toxin serotypesapparently have a lower potency and/or a shorter duration of activity ascompared to botulinum toxin type A. Clinical effects of peripheralintramuscular botulinum toxin type A are usually seen within one week ofinjection. The typical duration of symptomatic relief from a singleintramuscular injection of botulinum toxin type A averages about threemonths.

[0024] Although all the botulinum toxins serotypes apparently inhibitrelease of the neurotransmitter acetylcholine at the neuromuscularjunction, they do so by affecting different neurosecretory proteinsand/or cleaving these proteins at different sites. For example,botulinum types A and E both cleave the 25 kiloDalton (kD) synaptosomalassociated protein (SNAP-25), but they target different amino acidsequences within this protein. Botulinum toxin types B, D, F and G acton vesicle-associated protein (VAMP, also called synaptobrevin), witheach serotype cleaving the protein at a different site. Finally,botulinum toxin type C₁ has been shown to cleave both syntaxin andSNAP-25. These differences in mechanism of action may affect therelative potency and/or duration of action of the various botulinumtoxin serotypes. Significantly, it is known that the cytosol ofpancreatic islet B cells contains at least SNAP-25 (Biochem J 1;339 (pt1): 159-65 (April 1999)), and synaptobrevin (Mov Disord May 1995; 10(3):376).

[0025] The molecular weight of the botulinum toxin protein molecule, forall seven of the known botulinum toxin serotypes, is about 150 kD.Interestingly, the botulinum toxins are released by Clostridialbacterium as complexes comprising the 150 kD botulinum toxin proteinmolecule along with associated non-toxin proteins. Thus, the botulinumtoxin type A complex can be produced by Clostridial bacterium as 900 kD,500 kD and 300 kD forms. Botulinum toxin types B and C₁ is apparentlyproduced as only a 500 kD complex. Botulinum toxin type D is produced asboth 300 kD and 500 kD complexes. Finally, botulinum toxin types E and Fare produced as only approximately 300 kD complexes. The complexes (i.e.molecular weight greater than about 150 kD) are believed to contain anon-toxin hemaglutinin protein and a non-toxin and non-toxicnonhemaglutinin protein. These two non-toxin proteins (which along withthe botulinum toxin molecule comprise the relevant neurotoxin complex)may act to provide stability against denaturation to the botulinum toxinmolecule and protection against digestive acids when toxin is ingested.Additionally, it is possible that the larger (greater than about 150 kDmolecular weight) botulinum toxin complexes may result in a slower rateof diffusion of the botulinum toxin away from a site of intramuscularinjection of a botulinum toxin complex.

[0026] In vitro studies have indicated that botulinum toxin inhibitspotassium cation induced release of both acetylcholine andnorepinephrine from primary cell cultures of brainstem tissue.Additionally, it has been reported that botulinum toxin inhibits theevoked release of both glycine and glutamate in primary cultures ofspinal cord neurons and that in brain synaptosome preparations botulinumtoxin inhibits the release of each of the neurotransmittersacetylcholine, dopamine, norepinephrine, CGRP and glutamate.

[0027] Botulinum toxin type A can be obtained by establishing andgrowing cultures of Clostridium botulinum in a fermenter and thenharvesting and purifying the fermented mixture in accordance with knownprocedures. All the botulinum toxin serotypes are initially synthesizedas inactive single chain proteins which must be cleaved or nicked byproteases to become neuroactive. The bacterial strains that makebotulinum toxin serotypes A and G possess endogenous proteases andserotypes A and G can therefore be recovered from bacterial cultures inpredominantly their active form. In contrast, botulinum toxin serotypesC₁, D and E are synthesized by nonproteolytic strains and are thereforetypically unactivated when recovered from culture. Serotypes B and F areproduced by both proteolytic and nonproteolytic strains and thereforecan be recovered in either the active or inactive form. However, eventhe proteolytic strains that produce, for example, the botulinum toxintype B serotype only cleave a portion of the toxin produced. The exactproportion of nicked to unnicked molecules depends on the length ofincubation and the temperature of the culture. Therefore, a certainpercentage of any preparation of, for example, the botulinum toxin typeB toxin is likely to be inactive, possibly accounting for the knownsignificantly lower potency of botulinum toxin type B as compared tobotulinum toxin type A. The presence of inactive botulinum toxinmolecules in a clinical preparation will contribute to the overallprotein load of the preparation, which has been linked to increasedantigenicity, without contributing to its clinical efficacy.Additionally, it is known that botulinum toxin type B has, uponintramuscular injection, a shorter duration of activity and is also lesspotent than botulinum toxin type A at the same dose level.

[0028] High quality crystalline botulinum toxin type A can be producedfrom the Hall A strain of Clostridium botulinum with characteristics of≧3×10⁷ U/mg, an A₂₆₀/A₂₇₈ of less than 0.60 and a distinct pattern ofbanding on gel electrophoresis. The known Shantz process can be used toobtain crystalline botulinum toxin type A, as set forth in Shantz, E.J., et al, Properties and use of Botulinum toxin and Other MicrobialNeurotoxins in Medicine, Microbiol Rev. 56: 80-99 (1992). Generally, thebotulinum toxin type A complex can be isolated and purified from ananaerobic fermentation by cultivating Clostridium botulinum type A in asuitable medium. The known process can also be used, upon separation outof the non-toxin proteins, to obtain pure botulinum toxins, such as forexample: purified botulinum toxin type A with an approximately 150 kDmolecular weight with a specific potency of 1-2×10⁸ LD₅₀ U/mg orgreater; purified botulinum toxin type B with an approximately 156 kDmolecular weight with a specific potency of 1-2×10⁸ LD₅₀ U/mg orgreater, and; purified botulinum toxin type F with an approximately 155kD molecular weight with a specific potency of 1-2×10⁷ LD₅₀ U/mg orgreater.

[0029] Already prepared and purified botulinum toxins and toxincomplexes suitable for preparing pharmaceutical formulations can beobtained from List Biological Laboratories, Inc., Campbell, Calif.; theCentre for Applied Microbiology and Research, Porton Down, U.K.; Wako(Osaka, Japan), as well as from Sigma Chemicals of St Louis, Mo.

[0030] Pure botulinum toxin is so labile that it is generally not usedto prepare a pharmaceutical composition. Furthermore, the botulinumtoxin complexes, such a the toxin type A complex are also extremelysusceptible to denaturation due to surface denaturation, heat, andalkaline conditions. Inactivated toxin forms toxoid proteins which maybe immunogenic. The resulting antibodies can render a patient refractoryto toxin injection.

[0031] As with enzymes generally, the biological activities of thebotulinum toxins (which are intracellular peptidases) is dependant, atleast in part, upon their three dimensional conformation. Thus,botulinum toxin type A is detoxified by heat, various chemicals surfacestretching and surface drying. Additionally, it is known that dilutionof the toxin complex obtained by the known culturing, fermentation andpurification to the much, much lower toxin concentrations used forpharmaceutical composition formulation results in rapid detoxificationof the toxin unless a suitable stabilizing agent is present. Dilution ofthe toxin from milligram quantities to a solution containing nanogramsper milliliter presents significant difficulties because of the rapidloss of specific toxicity upon such great dilution. Since the toxin maybe used months or years after the toxin containing pharmaceuticalcomposition is formulated, the toxin must be stabilized with astabilizing agent. The only successful stabilizing agent for thispurpose has been the animal derived proteins albumin and gelatin. And asindicated, the presence of animal derived proteins in the finalformulation presents potential problems in that certain stable viruses,prions or other infectious or pathogenic compounds carried through fromdonors can contaminate the toxin.

[0032] Furthermore, any one of the harsh pH, temperature andconcentration range conditions required to lyophilize (freeze-dry) orvacuum dry a botulinum toxin containing pharmaceutical composition intoa toxin shipping and storage format (ready for use or reconstitution bya physician) can detoxify the toxin. Thus, animal derived or donor poolproteins such as gelatin and serum albumin have been used with somesuccess to stabilize botulinum toxin.

[0033] A commercially available botulinum toxin containingpharmaceutical composition is sold under the trademark BOTOX® (availablefrom Allergan, Inc., of Irvine, Calif.). BOTOX® consists of a purifiedbotulinum toxin type A complex, albumin and sodium chloride packaged insterile, vacuum-dried form. The botulinum toxin type A is made from aculture of the Hall strain of Clostridium botulinum grown in a mediumcontaining N-Z amine and yeast extract. The botulinum toxin type Acomplex is purified from the culture solution by a series of acidprecipitations to a crystalline complex consisting of the active highmolecular weight toxin protein and an associated hemagglutinin protein.The crystalline complex is re-dissolved in a solution containing salineand albumin and sterile filtered (0.2 microns) prior to vacuum-drying.BOTOX® can be reconstituted with sterile, non-preserved saline prior tointramuscular injection. Each vial of BOTOX® contains about 100 units(U) of Clostridium botulinum toxin type A purified neurotoxin complex,0.5 milligrams of human serum albumin and 0.9 milligrams of sodiumchloride in a sterile, vacuum-dried form without a preservative.

[0034] To reconstitute vacuum-dried BOTOX® sterile normal saline withouta preservative; 0.9% Sodium Chloride Injection is used by drawing up theproper amount of diluent in the appropriate size syringe. Since BOTOX®is denatured by bubbling or similar violent agitation, the diluent isgently injected into the vial. BOTOX® should be administered within fourhours after reconstitution. During this time period, reconstitutedBOTOX® is stored in a refrigerator (2° to 8° C.). Reconstituted BOTOX®is clear, colorless and free of particulate matter. The vacuum-driedproduct is stored in a freezer at or below −5° C. BOTOX® is administeredwithin four hours after the vial is removed from the freezer andreconstituted. During these four hours, reconstituted BOTOX® can bestored in a refrigerator (2° to 8° C.). Reconstituted BOTOX® is clear,colorless and free of particulate matter.

[0035] It has been reported that botulinum toxin type A has been used inclinical settings as follows:

[0036] (1) about 75-125 units of BOTOX® per intramuscular injection(multiple muscles) to treat cervical dystonia;

[0037] (2) 5-10 units of BOTOX® per intramuscular injection to treatglabellar lines (brow furrows) (5 units injected intramuscularly intothe procerus muscle and 10 units injected intramuscularly into eachcorrugator supercilii muscle);

[0038] (3) about 30-80 units of BOTOX® to treat constipation byintrasphincter injection of the puborectalis muscle;

[0039] (4) about 1-5 units per muscle of intramuscularly injected BOTOX®to treat blepharospasm by injecting the lateral pre-tarsal orbicularisoculi muscle of the upper lid and the lateral pre-tarsal orbicularisoculi of the lower lid.

[0040] (5) to treat strabismus, extraocular muscles have been injectedintramuscularly with between about 1-5 units of BOTOX®, the amountinjected varying based upon both the size of the muscle to be injectedand the extent of muscle paralysis desired (i.e. amount of dioptercorrection desired).

[0041] (6) to treat upper limb spasticity following stroke byintramuscular injections of BOTOX® into five different upper limb flexormuscles, as follows:

[0042] (a) flexor digitorum profundus: 7.5 U to 30 U

[0043] (b) flexor digitorum sublimus: 7.5 U to 30 U

[0044] (c) flexor carpi ulnaris: 10 U to 40 U

[0045] (d) flexor carpi radialis: 15 U to 60 U

[0046] (e) biceps brachii: 50 U to 200 U. Each of the five indicatedmuscles has been injected at the same treatment session, so that thepatient receives from 90 U to 360 U of upper limb flexor muscle BOTOX®by intramuscular injection at each treatment session.

[0047] (7) to treat migraine, pericranial injected (injectedsymmetrically into glabellar, frontalis and temporalis muscles)injection of 25 U of BOTOX® has showed significant benefit as aprophylactic treatment of migraine compared to vehicle as measured bydecreased measures of migraine frequency, maximal severity, associatedvomiting and acute medication use over the three month period followingthe 25 U injection.

[0048] The success of botulinum toxin type A to treat a variety ofclinical conditions has led to interest in other botulinum toxinserotypes. A study of two commercially available botulinum type Apreparations (BOTOX® and Dysport®) and preparations of botulinum toxinstype B and F (both obtained from Wako Chemicals, Japan) has been carriedout to determine local muscle weakening efficacy, safety and antigenicpotential. Botulinum toxin preparations were injected into the head ofthe right gastrocnemius muscle (0.5 to 200.0 units/kg) and muscleweakness was assessed using the mouse digit abduction scoring assay(DAS). ED₅₀ values were calculated from dose response curves. Additionalmice were given intramuscular injections to determine LD₅₀ doses. Thetherapeutic index was calculated as LD₅₀/ED₅₀. Separate groups of micereceived hind limb injections of BOTOX® (5.0 to 10.0 units/kg) orbotulinum toxin type B (50.0 to 400.0 units/kg), and were tested formuscle weakness and increased water consumption, the later being aputative model for dry mouth. Antigenic potential was assessed bymonthly intramuscular injections in rabbits (1.5 or 6.5 ng/kg forbotulinum toxin type B or 0.15 ng/kg for BOTOX®). Peak muscle weaknessand duration were dose related for all serotypes. DAS ED₅₀ values(units/kg) were as follows: BOTOX®: 6.7, Dysport®: 24.7, botulinum toxintype B: 27.0 to 244.0, botulinum toxin type F: 4.3. BOTOX® had a longerduration of action than botulinum toxin type B or botulinum toxin typeF. Therapeutic index values were as follows: BOTOX®: 10.5, Dysport®:6.3, botulinum toxin type B: 3.2. Water consumption was greater in miceinjected with botulinum toxin type B than with BOTOX®, althoughbotulinum toxin type B was less effective at weakening muscles. Afterfour months of injections 2 of 4 (where treated with 1.5 ng/kg) and 4 of4 (where treated with 6.5 ng/kg) rabbits developed antibodies againstbotulinum toxin type B. In a separate study, 0 of 9 BOTOX® treatedrabbits demonstrated antibodies against botulinum toxin type A. DASresults indicate relative peak potencies of botulinum toxin type A beingequal to botulinum toxin type F, and botulinum toxin type F beinggreater than botulinum toxin type B. With regard to duration of effect,botulinum toxin type A was greater than botulinum toxin type B, andbotulinum toxin type B duration of effect was greater than botulinumtoxin type F. As shown by the therapeutic index values, the twocommercial preparations of botulinum toxin type A (BOTOX® and Dysport®)are different. The increased water consumption behavior observedfollowing hind limb injection of botulinum toxin type B indicates thatclinically significant amounts of this serotype entered the murinesystemic circulation. The results also indicate that in order to achieveefficacy comparable to botulinum toxin type A, it is necessary toincrease doses of the other serotypes examined. Increased dosage cancomprise safety. Furthermore, in rabbits, type B was more antigenic thanwas BOTOX®, possibly because of the higher protein load injected toachieve an effective dose of botulinum toxin type B. Eur J NeurolNovember 1999;6(Suppl 4):S3-S10.

[0049] Acetylcholine

[0050] Typically only a single type of small molecule neurotransmitteris released by each type of neuron in the mammalian nervous system. Theneurotransmitter acetylcholine is secreted by neurons in many areas ofthe brain, but specifically by the large pyramidal cells of the motorcortex, by several different neurons in the basal ganglia, by the motorneurons that innervate the skeletal muscles, by the preganglionicneurons of the autonomic nervous system (both sympathetic andparasympathetic), by the postganglionic neurons of the parasympatheticnervous system, and by some of the postganglionic neurons of thesympathetic nervous system. Essentially, only the postganglionicsympathetic nerve fibers to the sweat glands, the piloerector musclesand a few blood vessels are cholinergic as most of the postganglionicneurons of the sympathetic nervous system secret the neurotransmitternorepinephine. In most instances acetylcholine has an excitatory effect.However, acetylcholine is known to have inhibitory effects at some ofthe peripheral parasympathetic nerve endings, such as inhibition ofheart rate by the vagal nerve.

[0051] The efferent signals of the autonomic nervous system aretransmitted to the body through either the sympathetic nervous system orthe parasympathetic nervous system. The preganglionic neurons of thesympathetic nervous system extend from preganglionic sympathetic neuroncell bodies located in the intermediolateral horn of the spinal cord.The preganglionic sympathetic nerve fibers, extending from the cellbody, synapse with postganglionic neurons located in either aparavertebral sympathetic ganglion or in a prevertebral ganglion. Since,the preganglionic neurons of both the sympathetic and parasympatheticnervous system are cholinergic, application of acetylcholine to theganglia will excite both sympathetic and parasympathetic postganglionicneurons.

[0052] Acetylcholine activates two types of receptors, muscarinic andnicotinic receptors. The muscarinic receptors are found in all effectorcells stimulated by the postganglionic neurons of the parasympatheticnervous system, as well as in those stimulated by the postganglioniccholinergic neurons of the sympathetic nervous system. The nicotinicreceptors are found in the synapses between the preganglionic andpostganglionic neurons of both the sympathetic and parasympathetic. Thenicotinic receptors are also present in many membranes of skeletalmuscle fibers at the neuromuscular junction.

[0053] Acetylcholine is released from cholinergic neurons when small,clear, intracellular vesicles fuse with the presynaptic neuronal cellmembrane. A wide variety of non-neuronal secretory cells, such as,adrenal medulla (as well as the PC12 cell line) and pancreatic isletcells release catecholamines and parathyroid hormone, respectively, fromlarge dense-core vesicles. The PC12 cell line is a clone of ratpheochromocytoma cells extensively used as a tissue culture model forstudies of sympathoadrenal development. Botulinum toxin inhibits therelease of both types of compounds from both types of cells in vitro,permeabilized (as by electroporation) or by direct injection of thetoxin into the denervated cell. Botulinum toxin is also known to blockrelease of the neurotransmitter glutamate from cortical synaptosomescell cultures.

[0054] What is needed therefore is an effective, long lasting,non-surgical resection, non-radiotherapy, non-systemic drugadministration, therapeutic drug and method for treating parathyroiddisorders.

SUMMARY

[0055] The present invention meets this need and provides an effective,non-surgical resection, long term, non-radiotherapy, non-systemic drugadministration, therapeutic method for treating parathyroid disorders.

[0056] The drug within the scope of this invention for treatingparathyroid disorders is a neurotoxin. Significantly, the sameneurotoxin can be used to treat hyperparathyroidism, hypoparathyroidism,hypocalcemia and hypercalcemia depending upon factors such as the siteof local administration of the neurotoxin and the amount of neurotoxinto be administered.

[0057] As used herein “local administration” means direct injection of aneurotoxin into a parathyroid gland or into a sympathetic ganglion whichinnervates a parathyroid PTH secretory cell. Systemic routes ofadministration, such as oral and intravenous routes of administration,are excluded from the scope of “local administration” of a neurotoxin.

[0058] A method for treating a parathyroid disorder according to thepresent invention can be carried out by administration of atherapeutically effective amount of a neurotoxin to a patient, therebytreating the parathyroid disorder. The neurotoxin can administered to aparathyroid gland of the patient when the parathyroid disorder to betreated is hypoparathyroidism. Alternately, the neurotoxin can beadministered to a sympathetic ganglion which innervates a parathyroidPTH secreting cell when the parathyroid disorder to be treated ishyperparathyroidism.

[0059] A detailed method for treating a parathyroid disorder accordingto the present invention can comprise the step of administration of atherapeutically effective amount of a botulinum toxin to a patient.Thus, a method for treating hypoparathyroidism according to the presentinvention can comprise the step of local administration to a parathyroidgland (i.e. to one or more of the four normally present parathyroidglands) of a therapeutically effective amount of a botulinum toxin,thereby increasing a deficient parathyroid hormone secretion from aparathyroid cell which is capable of secreting parathyroid hormone, andeffectively treating the hypoparathyroidism. Furthermore, a methodwithin the scope of the present invention for treatinghyperparathyroidism, can comprise the step of local administration to asympathetic ganglion which innervates a parathyroid PTH secreting cellof a parathyroid gland of a therapeutically effective amount of abotulinum toxin, thereby reducing an excessive parathyroid hormonesecretion from the parathyroid cell and hence effectively treating thehyperparathyroidism.

[0060] The neurotoxin can be administered in an amount of between about10⁻³ U/kg and about 35 U/kg. 35 U/kg is an upper limit because itapproaches a lethal dose of certain neurotoxins, such as botulinum toxintype A. Other botulinum toxins, such as botulinum toxin type B, can besafely administered at several orders of magnitude higher dosage.Preferably, the neurotoxin is administered in an amount of between about10⁻² U/kg and about 25 U/kg. More preferably, the neurotoxin isadministered in an amount of between about 10⁻¹ U/kg and about 15 U/kg.Most preferably, the neurotoxin is administered in an amount of betweenabout 1 U/kg and about 10 U/kg. In many instances, an administration offrom about 0.1 units to about 300 units of a neurotoxin, such as abotulinum toxin type A, provides effective and long lasting therapeuticrelief. More preferably, from about 0.1 unit to about 100 units of aneurotoxin, such as a botulinum toxin type A, can be used and mostpreferably, from about 0.1 unit to about 50 units of a neurotoxin, suchas a botulinum toxin type A, can be locally administered into a targettissue such as the thyroid or a sympathetic ganglion with efficaciousresults. In a particularly preferred embodiment of the presentinvention, a parathyroid gland or a sympathetic ganglion whichinnervates a parathyroid gland, can be locally administered with fromabout 1 unit to about 20 units of a neurotoxin (such as botulinum toxintype A) to achieve therapeutically effective results.

[0061] The neurotoxin can be made by a Clostridial bacterium, such as bya Clostridium botulinum, Clostridium butyricum, Clostridium beratti orClostridium tetani bacterium. Additionally, the neurotoxin can be amodified neurotoxin, that is a neurotoxin that has at least one of itsamino acids deleted, modified or replaced, as compared to the native orwild type neurotoxin. Furthermore, the neurotoxin can be a recombinantproduced neurotoxin or a derivative or fragment thereof.

[0062] The neurotoxin can be a botulinum toxin, such as one of thebotulinum toxin serotypes A, B, C₁, D, E, F or G. Preferably, theneurotoxin is botulinum toxin type A and the neurotoxin is locallyadministered by direct injection of the neurotoxin into a parathyroidgland or into a sympathetic ganglion which innervates the parathyroidgland.

[0063] A detailed embodiment of a method within the scope of the presentinvention for treating a parathyroid disorder can comprise the step ofinjecting a therapeutically effective amount of a botulinum toxin into aparathyroid gland of a human patient, thereby increasing a parathyroidhormone (PTH) secretion from a parathyroid hormone secreting parathyroidcell of the parathyroid gland and treating a parathyroid disorder.

[0064] Another detailed embodiment of a method within the scope of thepresent invention for treating a parathyroid disorder of a human patientcan comprise the step of local administration to a cholinergicinfluenced parathyroid PTH secreting cell of a parathyroid gland of ahuman patient of a therapeutically effective amount of botulinum toxintype A, thereby increasing a cholinergic influenced deficientparathyroid hormone secretion from the parathyroid cell of theparathyroid gland and treating the parathyroid disorder.

[0065] Another method within the scope of the present invention is amethod for treating a parathyroid disorder by administration of aneurotoxin to a sympathetic nervous system of a patient. In this methodthe neurotoxin is locally administered to a sympathetic ganglion whichinnervates a parathyroid hormone secreting parathyroid cell and theparathyroid disorder is hyperparathyroidism.

[0066] A detailed embodiment of a method within the scope of the presentinvention for treating a parathyroid disorder of a human patient cancomprise the step of in vivo, local administration to a sympatheticganglion, which innervates a parathyroid hormone secreting prothyroidcell of a parathyroid gland of a patient, of a therapeutically effectiveamount of a botulinum toxin, thereby decreasing an excessive parathyroidhormone secretion from the parathyroid cell of the parathyroid gland andtreating hyperparathyroidism.

[0067] A detailed embodiment of the present invention is a method fortreating a parathyroid disorder by injecting a therapeutically effectiveamount of a botulinum toxin into a parathyroid gland of a human patient,thereby increasing a secretion of a parathyroid hormone from aparathyroid cell and treating the parathyroid disorder. Preferably, thesecretion treated is a cholinergic influenced secretion and thebotulinum toxin used is botulinum toxin type A, although the botulinumtoxin can selected from the group consisting of botulinum toxin types A,B, C (i.e. C₁), D, E, F and G.

[0068] My invention also includes within its scope, a method fortreating hypocalcemia, the method comprising the step of localadministration to a parathyroid hormone secreting parathyroid cell of aparathyroid gland of a therapeutically effective amount of a botulinumtoxin, thereby increasing a deficient parathyroid hormone secretion fromthe parathyroid cell and treating hypocalcemia. Additionally, myinvention also includes within its scope a method for treatinghypercalcemia, the method comprising the step of local administration toa sympathetic ganglion which innervates a parathyroid hormone secretingparathyroid cell of a parathyroid gland of a therapeutically effectiveamount of a botulinum toxin, thereby decreasing an excessive parathyroidhormone secretion from the parathyroid cell of the parathyroid gland andtreating hypercalcemia.

DESCRIPTION

[0069] The present invention is based upon the discovery that aparathyroid disorder can be treated by in vivo administration of aneurotoxin to a patient. Thus administration of a neurotoxin to aparathyroid gland of a patient can remove an inhibitory cholinergiceffect upon parathyroid hormone secretion by the parathyroid gland,thereby providing an effective treatment for hypoparathyroidism and/orhypocalcemia. Additionally, administration of a neurotoxin to asympathetic ganglion which innervates a parathyroid hormone secretorycell of a parathyroid gland can remove a stimulatory adrenergic effectupon parathyroid hormone secretion, thereby providing an effectivetreatment for hyperparathyroidism and/or hypercalcemia.

[0070] Thus, parathyroid disorders can be treated, according to thepresent invention, by the alternative therapies of (a) localadministration of a neurotoxin to one or more of the parathyroid glands,or; (b) local administration of a neurotoxin to a parathyroid glandinnervating sympathetic ganglion of a patient, thereby resulting in,respectively, an increase of a secretion from a parathyroid cell, or adecrease in a secretion from a parathyroid chief (PTH secretory capable)cell

[0071] I have discovered that a particular neurotoxin, botulinum toxin,can be used with dramatic ameliorative effect to treat a parathyroiddisorder, thereby significantly superseding thereby current therapeuticregimens, such as surgical removal of parathyroid gland tissue to treathyperparathyroidism and calcium supplementation to treathypoparathyroidism.

[0072] Significantly, a single local administration of a neurotoxin,such as a botulinum toxin to one or more of the parathyroid glands,according to the present invention, can increase parathyroid hormonesecretion and thereby treat symptoms of hypoparathyroidism. I have alsodiscovered that a single local administration of a neurotoxin, such as abotulinum toxin to one or more of the sympathetic ganglia whichinnervate a parathyroid gland, according to the present invention, canreduce parathyroid hormone secretion and thereby treat symptoms ofhyperparathyroidism. In either case, the symptoms of the parathyroiddisorder can be alleviated for at least about from 2 months to about 6months per neurotoxin administration. Notably, it has been reported thatglandular tissue treated by a botulinum toxin can show a reducedsecretory activity for as long as 27 months post injection of the toxin.Laryngoscope 1999; 109:1344-1346, Laryngoscope 1998;108:381-384. Myinvention also includes within its scope the use of an implantedsustained release neurotoxin complex so as to provide therapeutic relieffrom a chronic parathyroid disorder. Thus, the neurotoxin can beimbedded within, absorbed, or carried by a suitable polymer matrix whichcan be implanted or embedded in or on a parathyroid gland or sympatheticganglion so as to provide a year or more of delayed and controlledrelease of the neurotoxin to the desired target tissue.

[0073] The hypoparathyroidism treatable by the present invention isprimary hypoparathyroidism. Secondary hypoparathyroidism is nottreatable by the present invention, because the present invention isbased upon a therapeutic, local administration of a neurotoxin to one ormore of the parathyroid glands and/or to a sympathetic ganglion whichinnervates the one or more of the parathyroid glands. Additionally theprimary hypoparathyroidism treatable by the present invention ishypoparathyroidism which has as a causative factor the inhibitoryactivity upon parathyroid hormone secretion of cholinergic,parasympathetic innervation of the parathyroid.

[0074] Similarly, the hyperparathyroidism treatable by the presentinvention is primary hyperparathyroidism. Secondary hyperparathyroidismis not treatable by the present invention, because the present inventionis based upon a therapeutic, local administration of a neurotoxin to oneor more of the parathyroid glands and/or to a sympathetic ganglion whichinnervates the one or more of the parathyroid glands. Additionally theprimary hyperparathyroidism treatable by the present invention ishyperparathyroidism which has as a causative factor the stimulatoryactivity upon parathyroid hormone secretion of sympathetic innervationof the parathyroid.

[0075] Notably, hypoparathyroidism resulting from a combination offactors, including inhibitory parasympathetic activity, is treatable bya method within the scope of the present invention. Similarly,hyperparathyroidism resulting from a combination of factors, includingstimulatory sympathetic activity, is treatable by a method within thescope of the present invention.

[0076] Local Administration of a Neurotoxin to the Parathyroid

[0077] A preferred embodiment of the present invention is to inject aparathyroid gland of a patient with from 0.1 to 100 units, morepreferably from 1 to 50 units, and most preferably from 1 to 10 units ofa neurotoxin (such as a botulinum toxin type A), to thereby cause anincrease of parathyroid hormone secretion. The present invention alsoincludes within its scope treatment of a parathyroid disorder due tohyperplasic, hypertonic or hypertrophic parathyroid chief cells. Aparathyroid disorder can be effectively treated by local administrationof a neurotoxin, such as for example 0.1 to 100 units of botulinum toxintype A, to cholinergic, postganglionic, parasympathetic neurons whichinnervate the dysfunctional, parathyroid cells. Without wishing to bebound by theory, the botulinum toxin is believed to act to increaseparathyroid hormone secretion by inhibiting release of acetylcholineneurotransmitter from cholinergic, postganglionic parasympathetic fiberswhich provide inhibitory innervation of parathyroid PTH secreting cells.

[0078] A neurotoxin, such as a botulinum toxin, locally administered invivo to the parathyroid to thereby remove an inhibitory effect upon asecretory activity of a parathyroid hormone secreting parathyroid cell.The parathyroid hormone secreting parathyroid cell is cholinergicallyinnervated such that the proteolytic light chain of the toxin isinternalized by a cholinergic neuron which inhibitoraly influences asecretory activity of the parathyroid hormone secreting parathyroidcell.

[0079] Thus, cholinergically innervated parathyroid cells can be treatedby local administration of a neurotoxin, such as a botulinum toxin. Bylocal administration, of a parathyroid gland, it is meant that theneurotoxin is administered directly to or to the immediate vicinity ofthe parathyroid tissue to be treated.

[0080] The specific dosage appropriate for administration is readilydetermined by one of ordinary skill in the art according to the factordiscussed above. The dosage can also depend upon the size of theparathyroid tissue mass to be treated or denervated, and the commercialpreparation of the toxin. Additionally, the estimates for appropriatedosages in humans can be extrapolated from determinations of the amountsof botulinum required for effective denervation of other tissues. Thus,the amount of botulinum A to be injected is proportional to the mass andlevel of activity of the thyroid tissue to be treated. Generally,between about 0.01 and 35 units per kg of patient weight of a botulinumtoxin, such as botulinum toxin type A, can be administered toeffectively accomplish a toxin induced parathyroid tissue secretion downregulation upon administration of the neurotoxin into the thyroid. Lessthan about 0.01 U/kg of a botulinum toxin does not have a significanttherapeutic effect upon the secretory activity of a parathyroid cell,while more than about 35 U/kg of a botulinum toxin approaches a toxicdose the neurotoxin. Careful placement of the injection needle and a lowvolume of neurotoxin used prevents significant amounts of botulinumtoxin from appearing systemically. A more preferred dose range is fromabout 0.01 U/kg to about 25 U/kg of a botulinum toxin, such as thatformulated as BOTOX®. The actual amount of U/kg of a botulinum toxin tobe administered depends upon factors such as the extent (mass) and levelof activity of the thyroid tissue to be treated and the administrationroute chosen. Botulinum toxin type A is a preferred botulinum toxinserotype for use in the methods of the present invention.

[0081] Diagnostic aids to determine hyper or hypoparathyroidism, bylocalization of the dysfunctional parathyroid tissue, includethallium-201/technetium-99m subtraction scintigraphy, computedtomography, ultrasonography, and magnetic resonance. Localizationstudies help by suggesting the most direct approach to the abnormalparathyroid tissue including whether surgery begins with a neck ormediastinal exploration. Particular scintigraphic techniques used tolocalize hyper or hypoplasic parathyroid glands, have included acombination of radiotracers including thallium-201 chloride ortechnetium-99m sestamibi and technetium-99m-sodium pertechnetate oriodine-123 sodium iodide. The processing of digital images obtainedincludes background correction and subtraction of the parathyroid imagefrom the composite parathyroid and parathyroid image. These methodsexploit the different biological behavior and tissue distribution of thetwo tracers, namely the parathyroid specific pertechnetate or iodineuptake relative to the more diffuse perfusion dependent distribution ofthallium or sestamibi.

[0082] It has been reported that the neuronal selectivity of clostridialneurotoxins is a result of a very selective binding and cell entrymechanism. Although a site of action of botulinum toxin is theneuromuscular junction, where the toxin binds rapidly and prevents therelease of acetylcholine from cholinergic neurons, it is known thatclostridial neurotoxins are able to enter certain neurosecretory cells(for example PC12 cells) via a low affinity receptor if highconcentrations of the neurotoxin are incubated with the cells forprolonged periods. This process appears to use a pathway via a receptorwhich is distinct from the highly specific and high affinity receptorpresent at the neuromuscular junction. Additionally, it has beenreported that certain clostridial toxins have effects on phagocytecells, such as macrophages, where entry into the cell is presumed to bevia the specific phagocytic activity of these cells. Furthermore,incubation of certain adipocytes (i.e. fat cells) with botulinum toxintype A has been reported to inhibit glucose uptake by the adipocytes.The mechanism of the glucose uptake inhibition is apparently due totoxin inhibition of plasma membrane fusion or docking of cytosolic,recyclable membrane vesicles (RMVs), the RMVs containing glucosetransporter proteins. PCT publication WO 94/21300.

[0083] Thus, while it is known that the botulinum toxins have a knownbinding affinity for cholinergic, pre-synaptic, peripheral motorneurons, it has been reported that botulinum toxins can also bind to andtranslocate into a variety of non-neuronal secretory cells, where thetoxin then acts, in the known manner, as an endoprotease upon itsrespective secretory vessel-membrane docking protein. Because of therelatively lower affinity of the botulinum toxins for secretory cells,such as parathyroid cells, as compared to the affinity of the botulinumtoxin for the cholinergic neurons which innervate parathyroid chiefcells, the botulinum toxin can be injected into secretory or glandulartissues to provide a high local concentration of the toxin, therebyfacilitating effect of the toxin upon both cholinergic neuron anddirectly upon parathyroid secretory cell. Thus, the present invention isapplicable to the treatment of parathyroid disorders in circumstanceswhere the target parathyroid secretory cells have little or nocholinergic innervation. Local administration of a neurotoxin to theparathyroids at a high dose level is carried out to treat primaryhyperparathyroidism.

[0084] Preferably, a neurotoxin used to practice a method within thescope of the present invention is a botulinum toxin, such as one of theserotype A, B, C, D, E, F or G botulinum toxins. Preferably, thebotulinum toxin used is botulinum toxin type A, because of its highpotency in humans, ready availability, and known safe and efficacioususe for the treatment of skeletal muscle and smooth muscle disorderswhen locally administered by intramuscular injection.

[0085] The present invention includes within its scope the use of anyneurotoxin which has a long duration therapeutic effect when locallyapplied to treat a parathyroid cell disorder of a patient. For example,neurotoxins made by any of the species of the toxin producingClostridium bacteria, such as Clostridium botulinum, Clostridiumbutyricum, and Clostridium beratti can be used or adapted for use in themethods of the present invention. Additionally, all of the botulinumserotypes A, B, C, D, E, F and G can be advantageously used in thepractice of the present invention, although type A is the most preferredserotype, as explained above. Practice of the present invention canprovide effective relief of a parathyroid disorder for from 2-27 monthsor longer in humans from a single injection of the neurotoxin.

[0086] Botulinum toxin is believed to be able to block the release ofany vesicle mediated exocytosis from any secretory (i.e. neuronal,glandular, secretory, chromaffin) cell type, as long as the light chainof the botulinum toxin is translocated into the intracellular medium.For example, the intracellular protein SNAP-25 is widely distributed inboth neuronal and non-neuronal secretory cells and botulinum toxin typeA is an endopeptidase for which the specific substrate is SNAP-25. Thus,while cholinergic neurons have a high affinity acceptor for thebotulinum and tetanus toxins (and are therefore more sensitive thanother neurons and other cells to the inhibition of vesicle mediatedexocytosis of secretory compounds), as the toxin concentration israised, non-cholinergic sympathetic neurons, chromaffin cells and othercell types can take up a botulinum toxin and show reduced exocytosis.

[0087] Hence, by practice of the present disclosed invention secretoryparathyroid cells with little or no cholinergic innervation can betreated by use of an appropriately higher concentration of a botulinumtoxin to bring about therapeutic relief from a parathyroid disorder suchas primary hyperparathyroidism. Local administration to a parathyroidgland of a lower dose neurotoxin (i.e. from about 1 unit to about 20units of botulinum toxin type A per parathyroid gland) can be used toteat hypoparathyroidism, as previously set forth.

[0088] Local Administration of a Neurotoxin to a Sympathetic Ganglion

[0089] Significantly, a method within the scope of the present inventionfor reducing an excessive parathyroid hormone secretion from aparathyroid gland comprises the step of local administration of aneurotoxin to the sympathetic nervous system. Sympathetic innervation ofthe parathyroid is know to exist. Thus, sympathetic nerve fibers caninhibit parathyroid hormone secretion by acting via adrenergic receptorson parathyroid hormone secreting parathyroid cells. A method within thescope of the present invention can therefore be carried out by localadministration of a neurotoxin to a preganglionic sympathetic (i.e.cholinergic) neuron which innervates a parathyroid cell. Thecholinergic, preganglionic, sympathetic neuron synapses with adrenergic,post-ganglionic, sympathetic fibers, and these later sympathetic neuronshave a stimulatory effect upon parathyroid hormone secretion byparathyroid gland chief cells. Preferably, the sympathetic ganglion towhich a neurotoxin is administered, according to the preset invention,is a cervical ganglion.

[0090] Cervical ganglion block according to the present invention can becarried out in the same manner as a celiac plexus block. Thus, theneurolytic celiac plexus block is a known procedure for treatingintractable pain resulting from upper abdominal viscera cancer. RegAnest Pain Med 1998; 23(1):37-48. Thus, it is known to inject the celiacplexus with ethanol or phenol to provide relief from the pain which canresult from pancreatic cancer or from pancreatitis. AJG1999;94(4):872-874. Hence, an antinociceptive injection of the cervicalganglia can be carried out as by either a percutaneous procedure or asan open (intraoperative) injection. The percutaneous (closed) procedurecan be carried out using an anterior approach using a very thin needle(22 Gauge). Cervical ganglion block is preferably carried out withcomputed tomography (CT) (as opposed to fluoroscopic) needle guidance,using a single thin needle.

[0091] Furthermore, a method within the scope of the present inventioncan provide improved patient function. “Improved patient function” canbe defined as an improvement measured by factors such as a reduced pain,reduced time spent in bed, increased ambulation, healthier attitude,more varied lifestyle and/or healing permitted by normal muscle tone.Improved patient function is synonymous with an improved quality of life(QOL). QOL can be assesses using, for example, the known SF-12 or SF-36health survey scoring procedures. SF-36 assesses a patient's physicaland mental health in the eight domains of physical functioning, rolelimitations due to physical problems, social functioning, bodily pain,general mental health, role limitations due to emotional problems,vitality, and general health perceptions. Scores obtained can becompared to published values available for various general and patientpopulations.

[0092] As set forth above, I have discovered that a surprisinglyeffective and long lasting therapeutic effect can be achieved by localadministration of a neurotoxin to a parathyroid or to a sympatheticganglion which innervates a parathyroid hormone secreting parathyroidcell of a human patient. In its most preferred embodiment, the presentinvention is practiced by direct injection into a parathyroid gland, orinto a sympathetic ganglion which innervates a parathyroid gland, of atherapeutically effective amount of botulinum toxin, such as botulinumtoxin type A. It has been reported that at the neuroglandular junction,the chemical denervation effect of a single injection of botulinumtoxin, such as botulinum toxin type A, has a duration of action of up to27 months.

[0093] The present invention includes within its scope: (a) neurotoxincomplex as well as pure neurotoxin obtained or processed by bacterialculturing, toxin extraction, concentration, preservation, freeze dryingand/or reconstitution and; (b) modified or recombinant neurotoxin, thatis neurotoxin that has had one or more amino acids or amino acidsequences deliberately deleted, modified or replaced by knownchemical/biochemical amino acid modification procedures or by use ofknown host cell/recombinant vector recombinant technologies, as well asderivatives or fragments of neurotoxins so made, and includesneurotoxins with one or more attached targeting moieties for a cellsurface receptor present on a parathyroid hormone secreting parathyroidcell.

[0094] Botulinum toxins for use according to the present invention canbe stored in lyophilized or vacuum dried form in containers under vacuumpressure. Prior to lyophilization the botulinum toxin can be combinedwith pharmaceutically acceptable excipients, stabilizers and/orcarriers, such as albumin. The lyophilized or vacuum dried material canbe reconstituted with saline or water.

[0095] The route of administration and amount of a neurotoxin (such as abotulinum toxin serotype A, B, C, D, E, F or G) administered accordingto the present invention for treating a parathyroid disorder can varywidely according to various patient variables including size, weight,age, disease severity, responsiveness to therapy, and solubility anddiffusion characteristics of the neurotoxin toxin chosen. Furthermore,the extent of the parathyroid or ganglionic tissue influenced isbelieved to be proportional to the volume of neurotoxin injected, whilethe quantity of the denervation is, for most dose ranges, believed to beproportional to the concentration of neurotoxin injected.

[0096] Methods for determining the appropriate route of administrationand dosage are generally determined on a case by case basis by theattending physician. Such determinations are routine to one of ordinaryskill in the art (see for example, Harrison's Principles of InternalMedicine (1998), edited by Anthony Fauci et al., 14^(th) edition,published by McGraw Hill). For example, to treat a parathyroid disorder,a solution of botulinum toxin type A complex can be endoscopically orintraperitoneally injected directly into the tissues of the parathyroid,thereby substantially avoiding entry of the toxin into the systemiccirculation.

EXAMPLES

[0097] The following examples provide those of ordinary skill in the artwith specific preferred methods within the scope of the presentinvention for carrying out the present invention and are not intended tolimit the scope of what the inventor regards as his invention. In eachof the following examples, the specific amount of a botulinum toxinadministered depends upon a variety of factors to be weighed andconsidered within the discretion of the attending physician and in eachof the examples insignificant amounts of botulinum toxin enter appearsystemically with no significant side effects. Units of botulinum toxininjected per kilogram (U/kg) below are per kg of total patient weight.For example, 3 U/kg for a 70 kg patient calls for an injection of 210units of the botulinum toxin.

Example 1 Intraoperative Administration of Neurotoxin

[0098] Intraoperative, local administration of a neurotoxin to aparathyroid can be carried out as follows. The procedure can beperformed under general endotracheal anesthesia. The patient's neck canbe extended by inflating a pillow or inserting a thyroid roll beneaththe shoulders. A symmetrical, low, collar incision can then be made inthe line of a natural skin crease approximately 1 to 2 cm above theclavicle. The incision can be carried through the skin, subcutaneoustissue, and platysma muscle down to the dense cervical fascia thatoverlies the strap muscles and anterior jugular veins. The upper flapcan then be raised to a level cephalad to the cricoid cartilage. Care istaken to avoid cutting sensory nerves. A small lower flap is alsoelevated to the level of the manubrial notch. Performing dissection ofthe flaps in the plane between the platysma muscle and the fasciaoverlying the strap muscles results in minimal bleeding. The cervicalfascia is then incised vertically in the midline.

[0099] Exposure of the parathyroid glands can generally be achieved byretracting the sternohyoid and sternothyroid muscles laterally Digitalor blunt dissection frees the parathyroids from the surrounding fascia.An exposed parathyroid gland can be directly injected with from 0.1 to50 units of a botulinum toxin, such as botulinum toxin type A. Careshould be taken to ensure that the parathyroid glands are not excised ordevascularized. Within one to seven days, parathyroid hormone secretionis substantially increased due to removal of cholinergic inhibition andthis effect persists for from about 2 to about 6 months.

Example 2 Local Administration of Neurotoxin to a Parathyroid

[0100] Local administration of a neurotoxin directly to or to thevicinity of a parathyroid gland can be accomplished by several methods.For example, by parathyroid endoscopy. An endoscope used for parathyroidtherapy can be modified to permit its use for direct injection of aneurotoxin, such as a botulinum toxin directly into parathyroid tissue.See for example U.S. Pat. No. 5,674,205. Once appropriately located, ahollow needle tip can be extended from the endoscope into parathyroidtissue and through which needle the neurotoxin can be injected into theparathyroid tissue of one or more of the parathyroid glands.

[0101] Additionally, fine needle aspiration for parathyroid biopsypurposes is known and can be used to inject a neurotoxin, rather than toaspirate parathyroid tissue. From 0.1 to 50 units of a botulinum toxin,such as botulinum toxin type A can thereby be injected into one or moreof the parathyroid glands. Within one to seven days, parathyroid hormonesecretion is substantially increased due to removal of cholinergicinhibition and this effect persists for from 2 to 6 months.

Example 3 Treatment of Hypoparathyroidism with Botulinum Toxin Type A

[0102] A 43 year old male is diagnosed with hypoparathyroidism. Betweenabout 0.1 U and about 50 U of a botulinum toxin type A preparation (forexample between about 0.1 units and about 50 units of BOTOX®) isinjected directly into one or more of the parathyroid glands, using oneof the techniques set forth in Examples 1 or 2 above. Within 1-7 daysthe symptoms of the hypoparathyroidism are alleviated and parathyroidhormone levels return to substantially normal levels. Alleviation of theparathyroid disorder persists for at least about 2 months to about 6months.

Example 4 Treatment of Hypoparathyroidism with Botulinum Toxin Type B

[0103] A 52 year old female is diagnosed with hypoparathyroidism.Between about 20 units and about 1000 units of a botulinum type Bpreparation is injected directly into one or more of the parathyroids,using one of the techniques set forth in Examples 1 or 2 above. Within1-7 days the symptoms of the hypoparathyroidism are alleviated.Parathyroid hormone levels return to substantially normal levels.Alleviation of the parathyroid disorder persists for at least about 2months to about 6 months.

Example 5 Treatment of Hypoparathyroidism with Botulinum Toxin Type C

[0104] A 58 year old female is diagnosed with hypoparathyroidism.Between about 10⁻³ U/kg and about 35 U/kg of a botulinum toxin type Cpreparation (for example between about 10 units and about 10,000 unitsof a botulinum type C preparation) is injected directly into one or moreof the parathyroids, using one of the techniques set forth in Examples 1or 2 above. Within 1-7 days the symptoms of the hypoparathyroidism arealleviated. Parathyroid hormone levels return to substantially normallevels. Alleviation of the parathyroid disorder persists for at leastabout 2 months to about 6 months.

Example 6 Treatment of Hypoparathyroidism with Botulinum Toxin Type D

[0105] A 56 year old obese female is diagnosed with hypoparathyroidism.Between about 10⁻³ U/kg and about 35 U/kg of a botulinum toxin type Dpreparation (for example between about 10 units and about 10,000 unitsof a botulinum type D preparation) is injected directly into one or moreof the parathyroids, using one of the techniques set forth in Examples 1or 2 above. Within 1-7 days the symptoms of the hypoparathyroidism arealleviated. Parathyroid hormone levels return to substantially normallevels. Alleviation of the parathyroid disorder persists for at leastabout 2 months to about 6 months.

Example 7 Treatment of Hypoparathyroidism with Botulinum Toxin Type E

[0106] A 61 year old female is diagnosed with hypoparathyroidism.Between about 10⁻³ U/kg and about 35 U/kg of a botulinum toxin type Epreparation (for example between about 10 units and about 10,000 unitsof a botulinum type E preparation) is injected directly into one or moreof the parathyroids, using one of the techniques set forth in Examples 1or 2 above. Within 1-7 days the symptoms of the hypoparathyroidism arealleviated. Parathyroid hormone levels return to substantially normallevels. Alleviation of the parathyroid disorder persists for at leastabout 2 months to about 6 months.

Example 8 Treatment of Hypoparathyroidism with Botulinum Toxin Type F

[0107] A 52 year old male is diagnosed with hypoparathyroidism. Betweenabout 10⁻³ U/kg and about 35 U/kg of a botulinum toxin type Fpreparation (for example between about 10 units and about 10,000 unitsof a botulinum type F preparation) is injected directly into one or moreof the parathyroids, using one of the techniques set forth in Examples 1or 2 above. Within 1-7 days the symptoms of the hypoparathyroidism arealleviated. Parathyroid hormone levels return to substantially normallevels. Alleviation of the parathyroid disorder persists for at leastabout 2 months to about 6 months.

Example 9 Treatment of Hypoparathyroidism with Botulinum Toxin Type G

[0108] A 59 year old female is diagnosed with hypoparathyroidism.Between about 10⁻³ U/kg and about 35 U/kg of a botulinum toxin type Gpreparation (for example between about 10 units and about 10,000 unitsof a botulinum type G preparation) is injected directly into one or moreof the parathyroids, using one of the techniques set forth in Examples 1or 2 above. Within 1-7 days the symptoms of the hypoparathyroidism arealleviated. Parathyroid hormone levels return to substantially normallevels. Alleviation of the parathyroid disorder persists for at leastabout 2 months to about 6 months.

Example 10 Treatment of Hyperparathyroidism with Botulinum Toxin Type A

[0109] A 27 year old female presents with symptoms of progressivelyworsening myalgias over the last six months. She was otherwiseasymptomatic and in good health. Physical examination is unremarkable.Family history and social history are likewise noncontributory. Aroutine screening serum chemistry profile reveals a serum calcium of12.0 mg/dl. Serum albumin, protein, magnesium, and chloride are allwithin normal limits. A parathormone assay level of 211 picograms/ml(normal=10 to 55 picograms/ml) is noted. Two subsequent calcium levelsand parathormone levels are persistently elevated and the diagnosis ofprimary hyperparathyroidism is made. A trial with atropine reduces theparathyroid hormone level.

[0110] Between about 10⁻³ U/kg and about 35 U/kg of a botulinum toxintype A preparation (for example between about 0.1 units and about 50units of BOTOX®) is injected directly into the cervical ganglia asfollows. A percutaneous procedure is carried out using an anteriorapproach with the patient in a supine position using a very thin needle(22 Gauge) with computed tomography needle guidance to reach thecervical ganglia. Within 1-7 days the symptoms of thehyperparathyroidism are alleviated. Parathyroid hormone levels return tonormal (are lowered). Alleviation of the parathyroid disorder persistsfor at least about 2 months to about 6 months.

Example 11 Treatment of Hyperparathyroidism with Botulinum Toxin TypesB-G

[0111] A 62 year old female presents with symptomatic hypercalcemia,serum calcium of 1-1.6 mg/dL above normal values, a creatinine clearancereduction greater than 30%, a 24-hr. urine calcium excretion >400 mg,and a bone mass <2 S.D.'s below normal. A diagnosis of primaryhyperparathyroidism is made. Between about 10⁻³ U/kg and about 35 U/kgof a botulinum toxin type B, C₁, D, E, F or G preparation (for examplebetween about 10 units and about 10,000 units of a botulinum type B-Gpreparation) is injected directly into the cervical ganglia as follows.A percutaneous procedure is carried out using an anterior approach withthe patient in a supine position using a very thin needle (22 Gauge)with computed tomography needle guidance to reach the cervical ganglia.Within 1-7 days the symptoms of the hyperparathyroidism are alleviatedand parathyroid hormone levels return to substantially normal levels.Alleviation of the hyperparathyroidism persists for at least about 2months to about 6 months.

Example 12 Treatment of Calcium Metabolism Disorders with BotulinumToxin Types A-G

[0112] A 28 year old female is diagnosed with hypocalcemia. Betweenabout 10⁻³ U/kg and about 35 U/kg of a botulinum toxin type A, B, C, D,E, F or G preparation (for example between about 0.1 units and about 50units of a botulinum toxin type A preparation) is injected directly intoone or more of the parathyroids using one of the techniques set forth inExamples 1 or 2 above. Within 1-7 days the symptoms of the hypocalcemiaare alleviated. Plasma calcium levels return to substantially normallevels. Alleviation of the hypocalcemia persists for at least about 2months to about 6 months.

[0113] Additionally, to treat hypercalcemia between about 10⁻³ U/kg andabout 35 U/kg of a botulinum toxin type A, B, C, D, E, F or Gpreparation (for example between about 0.1 units and about 50 units of abotulinum toxin type A preparation) is injected directly into thecervical ganglia as follows. A percutaneous procedure is carried outusing an anterior approach with the patient in a supine position using avery thin needle (22 Gauge) with computed tomography needle guidance toreach the cervical ganglia. Within 1-7 days the symptoms of thehypercalcemia are alleviated. Plasma calcium levels return to normal(are decreased). Alleviation of the hypercalcemia persists for at leastabout 2 to about 6 months.

[0114] Methods according to the invention disclosed herein has manyadvantages, including the following:

[0115] (1) the invention renders unnecessary many surgical proceduresfor effective treatment of a parathyroid disorder.

[0116] (2) systemic drug effects can be avoided by direct localapplication of a neurotoxin according to the present invention.

[0117] (3) the ameliorative effects of the present invention canpersists, on average, from about 2 months to about 6 months from asingle local administration of a neurotoxin as set forth herein.

[0118] Although the present invention has been described in detail withregard to certain preferred methods, other embodiments, versions, andmodifications within the scope of the present invention are possible.For example, a wide variety of neurotoxins can be effectively used inthe methods of the present invention. Additionally, the presentinvention includes local parathyroid administration methods wherein twoor more neurotoxins, such as two or more botulinum toxins, areadministered concurrently or consecutively. For example, botulinum toxintype A can be administered until a loss of clinical response orneutralizing antibodies develop, followed by administration of botulinumtoxin type E. Alternately, a combination of any two or more of thebotulinum serotypes A-G can be locally administered to control the onsetand duration of the desired therapeutic result. Furthermore,non-neurotoxin compounds can be administered prior to, concurrently withor subsequent to administration of the neurotoxin to proved adjuncteffect such as enhanced or a more rapid onset of denervation before theneurotoxin, such as a botulinum toxin, begins to exert its therapeuticeffect.

[0119] My invention also includes within its scope the use of aneurotoxin, such as a botulinum toxin, in the preparation of amedicament for the treatment of a parathyroid disorder by localadministration of the neurotoxin.

[0120] Accordingly, the spirit and scope of the following claims shouldnot be limited to the descriptions of the preferred embodiments setforth above.

I claim:
 1. A method for treating a parathyroid disorder, the methodcomprising the step of administration of a neurotoxin to a patient,thereby treating a parathyroid disorder.
 2. The method of claim 1 ,wherein the neurotoxin is administered to a parathyroid gland of thepatient.
 3. The method of claim 2 , wherein the parathyroid disordertreated is hypoparathyroidism.
 4. The method of claim 1 , wherein theneurotoxin is administered to a sympathetic ganglion which innervatesthe parathyroid.
 5. The method of claim 4 , wherein the parathyroiddisorder treated is hyperparathyroidism.
 6. The method of claim 1 ,wherein the neurotoxin is administered in an amount of between about10⁻³ U/kg and about 35 U/kg.
 7. The method of claim 1 , wherein theneurotoxin is made by a Clostridial bacterium.
 8. The method of claim 1, wherein the neurotoxin is a botulinum toxin.
 9. The method of claim 1, wherein the botulinum toxin is selected from the group consisting ofbotulinum toxin types A, B, C₁, D, E, F and G.
 10. The method of claim 1, wherein the neurotoxin is botulinum toxin type A.
 11. A method fortreating a parathyroid disorder, the method comprising the step ofadministration of a therapeutically effective amount of a botulinumtoxin to a patient, thereby treating a parathyroid disorder.
 12. Themethod of claim 11 , wherein the botulinum toxin is selected from thegroup consisting of botulinum toxin types A, B, C₁, D, E, F and G.
 13. Amethod for treating hypoparathyroidism, the method comprising the stepof local administration to a parathyroid gland of a therapeuticallyeffective amount of a botulinum toxin, thereby increasing a deficientparathyroid hormone secretion from a PTH secreting parathyroid cell andtreating hypoparathyroidism.
 14. The method of claim 13 , wherein thebotulinum toxin is selected from the group consisting of botulinum toxintypes A, B, C₁, D, E, F and G.
 15. A method for treatinghyperparathyroidism, the method comprising the step of localadministration to a sympathetic ganglion which innervates a PTHsecreting parathyroid cell of a therapeutically effective amount of abotulinum toxin, thereby reducing an excessive parathyroid hormonesecretion from the parathyroid cell and treating hyperparathyroidism.16. The method of claim 15 , wherein the botulinum toxin is selectedfrom the group consisting of botulinum toxin types A, B, C₁, D, E, F andG.
 17. A method for treating hypocalcemia, the method comprising thestep of local administration to a parathyroid gland of a therapeuticallyeffective amount of a botulinum toxin, thereby increasing a parathyroidhormone secretion from a parathyroid cell and treating hypocalcemia. 18.The method of claim 17 , wherein the botulinum toxin is selected fromthe group consisting of botulinum toxin types A, B, C₁, D, E, F and G.19. A method for treating hypercalcemia, the method comprising the stepof local administration to a sympathetic ganglion which innervates aparathyroid cell of a therapeutically effective amount of a botulinumtoxin, thereby decreasing a parathyroid hormone secretion from aparathyroid C cell and treating hypercalcemia.
 20. The method of claim19 , wherein the botulinum toxin is selected from the group consistingof botulinum toxin types A, B, C₁, D, E, F and G.